Today, semisynthetic .beta.-lactams such as Ampicillin, Amoxicillin, Cefaclor, Cephalexin, Cephadroxil and Cephaloglycin are prepared in industry by chemical methods, for example by reacting an amino .beta.-lactam such as 6-aminopenicillanic acid, usually having its carboxyl group protected, with an activated side chain derivative, followed by the removal of the protecting group by hydrolysis. It is important due to, for example, yield, that the amino .beta.-lactam, for example 6-APA, is used in a pure, dry form, preferably in a purity higher than 97%. For example, Ampicillin (6-D-.alpha.-aminophenylacetamidopenicillanic acid) can be prepared by reacting 6-APA, having a..suitable protected carboxyl group, with D-phenylglycine acidchloride, followed by removal of the protecting group by hydrolysis. These reactions typically involve costly steps such as sub zero degree Celcius conditions and organic solvents like methylene chloride and silylation reagents.
Enzymatic production of Ampicillin from pure 6-APA and a D-phenylglycine derivative (such as a lower alkyl ester) is known from West German patent application having publication No. 2,163,792, Austrian Patent No. 243,986, Dutch patent application No. 70-09138, West German patent application having publication No. 2,621,618 and European patent application having publication No. 339,751. Processes described in the prior art have typically used below 50 mM of the D-phenylglycine derivative and below 25 mM of 6-APA, the highest yield reported was 88% (European patent application having publication No. 339,751).
The amino .beta.-lactam such as 6-APA is commonly produced by enzymatic hydrolysis of a fermented -penicillin (for example penicillin V or penicillin G) followed by removal of the liberated side chain (phenoxyacetic acid etc.). Besides impurities originating from the fermentation, the resulting crude solution typically contains the amino .beta.-lactam at a concentration of 150-200 mM. The crude solution can be purified and crystallized to obtain pure 6-APA or 7-ADCA (in the 7-ADCA case, the fermented penicillin have been through another process before the hydrolysis step)..
The potential drawbacks of the known enzymatic methods for production of Ampicillin, Amoxicillin and Cephalexin (none have yet been upscaled to industrial applicability) are the high costs (yield losses) and investments due to the necessary unit operations incurred when the amino .beta.-lactam is isolated, purified and dried before being used as raw material for the reaction leading to a semisynthetic .beta.-lactam. Furthermore, the starting concentrations of the 6-APA are very low (typically less than 50 mM), thus making the isolation of the formed Ampicillin more difficult and thus more costly. Also, a higher yield in the enzymatic formation of Ampicillin is desirable.
A process for enzymatic synthesis of Amoxicillin is described in Agric. Biol. Chem. 44 (1980), 821 et seq., which process is performed in a reaction medium containing 2.5% (volume/volume) or more of 2-propanol and 5% (volume/volume) of other alcohols. When one of the last mentioned alcohols or 2.5% of 2-propanol are used, the initial concentration of the starting materials, D-.alpha.-(p-hydroxyphenyl)glycine methyl ester and 6-aminopenicillanic acid, is very low, i.e. 100 and 50 mM, respectively. When 5% of 2-propanol is used, the-initial concentration of the starting materials, D-.alpha.-(p-hydroxyphenyl)glycine methyl ester and 6-aminopenicillanic acid, is 460 and 230 mM, respectively. It is stated in this paper that the addition of more than 100 mM of D-.alpha.-(p-hydroxyphenyl)glycine methyl ester and of more than 50 mM of 6-aminopenicillanic acid markedly suppressed the ration of conversion of 6-aminopenicillanic acid into amoxicillin. The conclusion of this statement is that this publication teaches away for increasing the concentration of the amino .beta.-lactam and of the acylating agent in the reaction mixture.
After the effective filing date of the application for a patent on this invention, namely Sep. 18-21, 1990, a poster was published at a NATO Workshop. The poster dealt with the preparation of cephalosporins and, according to this poster, working at low temperature had several positive effects on the reaction. The highest concentration of acylating agent used by this work was 355 mM of D-.alpha.-phenylglycine methyl ester and there was no indication on the poster according to which it could be advantageous to use a higher concentration of the acylating agent.